Method of manufacturing a tissue-engineered prosthesis

ABSTRACT

Developing heart valves are exposed to dynamic strains by applying a dynamic pressure difference over the leaflets. The flow is kept to a minimum, serving only as a perfusion system, supplying the developing tissue with fresh nutrients. Standard heart valves were engineered based on B trileaflet scaffolds seeded with cells isolated from the human saphenous vein. Tissue compaction is constrained by the stent, inducing increasing pre-strain in the tissue. The dynamic strains the tissues are exposed to via the dynamic pressure difference, are estimated using finite element methods based on the mechanical properties of the neo-tissue, in order to get inside into the strain distribution over the leaflet.

CROSS-REFERENCE TO RELATED PATENT APPLICATION

This application is a Continuation of U.S. application Ser. No.11/816,271, filed Feb. 15, 2008, which is a National Stage applicationof PCT/EP2006/000877, filed Feb. 1, 2006, which claims priority onEuropean Patent Application No. 05003425.5, filed Feb. 17, 2005. Thecontents of the prior applications are incorporated by reference intheir entirety.

The invention is directed to a method of manufacturing atissue-engineered prosthesis having at least in an open condition a flowpassage, especially human heart valves. The invention is also directedto a respective bioreactor.

Such a method is known from WO2004/018008 A1 and WO2004/101012 A1. Thelatter also discloses a bioreactor for producing tissue prosthesis,particularly a heart valve.

Mechanical conditioning in a bioreactor profoundly affects thecomposition and structure and hence mechanical properties of tissueengineered heart valves. Up to now, the methods and bioreactorsdeveloped for culturing heart valves are flow-based and mimic the normalheart valve opening and closing behaviour. This method of conditioninghas resulted in enhanced tissue formation and mechanical properties,however, yet insufficient to serve as systemic pressure heart valvereplacement such as an aortic heart valve replacement.

In cardiovascular tissue engineering, stimulation of tissue formation bymechanical conditioning has proven to be a useful tool for developmentof functional cardiovascular structures enabling growth, repair, andremodeling. The main ways of conditioning a developing tissue are byapplying either flow or strain, or a combination of both. Variousbioreactors have been developed in the past years to apply specificconditioning protocols to growing cardiovascular structures.

Bioreactors that use flow as the main mechanical stimulus are, forexample, the bioreactors developed by Williams and Wick (2004) andNarita et al. (2004) to engineer blood vessels. Furthermore, a pulseduplicator system has been developed by Hoerstrup et al. (2000a,b) togrow heart valves as well as blood vessels, modified by Sodian et al.(2001, 2002b) to include the seeding procedure. Bioreactors that usestrains as the main mechanical cue are e.g. the bioreactors developed byNiklason et al. (1999, 2001) and Seliktar et al. (2000) for tissueengineering of blood vessels. In these bioreactors, the tissue isexposed to dynamic strains by applying intraluminal pulsatile pressuresvia an inflatable silicone tube. Niklason et al. (1999, 2001) applydynamic strains of about 5%, while Seliktar et al. (2000) use largerstrains of about 10%. For myocardial tissue, a comparable bioreactor isdeveloped by Gonen-Wadmany et al. (2004) applying dynamic strains(0-12%) to the developing tissue by pulsatile inflation of a siliconebulb, to which the tissue is attached. The flow applied to the tissuescultured in these strain-based bioreactors can be kept to a minimum andare mainly induced by medium circulation or the movement of the tissueitself. In another type of bioreactors, the physiological environment ofthe cardiovascular structure is mimicked, including both flow andstrain. These bioreactors can be used for testing native tissues, aswell as for tissue engineering and subsequent functionality testing. Abioreactor in which the physiological pressure waveforms, present in ablood vessel, can be applied is the system developed by McCulloch et al.(2004). A bioreactor in which physiological pressure waveforms can beapplied in combination with physiological flows is developed by Thompsonet al. (2002). As blood vessels are exposed to longitudinal strainsin-vivo as well, Mironov et al. (2003) developed a bioreactor thatexposes developing blood vessels to a physiological environmentincluding dynamic longitudinal strains. Hildebrand et al. (2004), Dumontet al. (2002), and Rutten et al. (2005) have developed bioreactors, inwhich the exact physiological conditions of a heart valve in-vivo can beapplied, with the latter providing the possibility to visualize valvefunction using MRI. Despite these efforts, the question still remains towhat extent the developing tissue should be exposed to mechanical cuesand which mechanical cues are optimal for tissue development.

The cells in the tissue engineered structures are responsible for theformation of the extracellular matrix and via mechanical conditioningthey can be stimulated to produce larger amounts of extracellularmatrix. Apart from the nature and magnitude of the mechanical cues, thecellular (pheno)type as well as the culture conditions (either 2D or ina 3D environment) affect the cellular responses to mechanical loading(Dethlefsen et al., 1996; Ueba et al., 1997; Watase et al. 1997; Kim etal. 1999; Chapman et al., 2000; O'Callaghan and Williams, 2000; Kim andMooney, 2000; Jockenhoevel et al., 2002; Lee et al., 2002; Engelmayr etal. 2005). Human saphenous vein cells were chosen as a cell source fortissue engineering of human heart valve leaflets. Not only do theyrepresent an easily accessible cell source, they were also shown to bemore sensitive to mechanical cues compared to human arterial derivedcells (Dethlefsen et al. 1996; Schnell et al., 2001). Although theoptimal conditioning procedure for this specific cell type still has tobe determined, increased extracellular matrix formation has beendemonstrated in engineered valve leaflet tissue-equivalents culturedwith these cells using dynamic strains (Mol et al., 2003). It is,therefore, hypothesized that the optimal conditioning protocol involvesdynamic strains to stimulate matrix production by the seeded vascularcells. An initial low shear stress environment combined with exposure ofthe developing tissue to physical stimuli has been suggested by Barronet al. (2003) to be advantageous for initial tissue development. Shearstresses, by application of flows, will most likely start to play asignificant role at a later stage when the leaflets are seeded withendothelial cells to provide a non-thrombogenic surface layer,stabilizing the underlying tissue prior to implantation (Nackman et al.,1998; Weston and Yoganathan, 2001).

It is the object of the present invention to improve the mechanicalproperties of tissue-engineered prosthesis.

The object is achieved by a method of manufacturing a tissue-engineeredprosthesis, having at least in an open condition a flow passage,especially human heart valves, comprising the steps of:

-   -   Placing a seeded 3D scaffold in a bioreactor chamber    -   Providing at least one perfusion flow of a nutrient medium in        the bioreactor chamber to supply said seeded 3D scaffold and/or        a thereon developing tissue with nutrients whereby a flow        passage of the seeded 3D scaffold and/or the thereon developing        tissue in relation to the flow passage of the finished        prosthesis is restricted or zero;    -   Applying in addition a dynamic pressure difference over the 3D        scaffold and/or thereon developing tissue, depending on the        condition and/or stage of tissue development to create strain in        the 3D scaffold and/or thereon developing tissue; and    -   opening the restricted or zero flow passage to its finished        size.

Contrary to the former flow-based methods, the present inventionprovides for a strain-based approach. In order to develop sufficientstrain in the 3D scaffold and/or thereon developing tissue thedeveloping prosthesis is exposed to dynamic strains by applying adynamic pressure difference. The strain-based approach has shown torender tissue-engineered prosthesis with superior tissue formation andorganisation and hence improved mechanical properties. The appropriatestrain can be provided by restricting or closing the flow passage of thedeveloping prosthesis during the duration stay in the bioreactorchamber. The restriction or closure of the flow passage during tissuedevelopment is opened to its final size as usual for the respectiveprosthesis, especially human heart valve. For example, only a smallopening could be present allowing only for a minimum perfusion flow of anutrition medium from the one side of the prosthesis to the other. Incase the flow passage is completely closed two separate perfusion flowscould be provided, one on each side of the developing prosthesis. Withheart valves the restriction may be achieved by coaptation of theleaflets.

The goal of one embodiment is to set up a basic concept to engineerhuman heart valve leaflets using dynamic strains in combination withminimized flows to stimulate extracellular matrix formation. This is anovel approach as all currently used conditioning approaches for tissueengineering of heart valves concentrate on the application of pulsatileflows, with none or limited tissue straining. For this purpose, a newbioreactor system is developed, the Diastolic Pulse Duplicator (DPD), todeliver dynamic strains to the heart valve leaflets by applying adynamic pressure difference over the closed leaflets. Requirements forsuch a bioreactor system are: 1) simplicity in its use, 2) sized smallto save on culture medium and incubator space, 3) usage of biocompatiblematerials, 4) maintenance of sterility over prolonged periods of time,and 5) the ability to monitor and control the applied transvalvularpressure. As a stented valve geometry is used in this study,compaction-induced prestrain develops in the leaflets as tissuecompaction, common in growing and healing tissues, is constrained by therigid stent. Medium is circulating in the DPD at low speed (4 ml/min) toprovide oxygen and fresh nutrients to the developing tissue and toremove waste products.

The feasibility of the strain-based approach for human heart valvetissue engineering is demonstrated. Tissue formation and mechanicalproperties of leaflets exposed to dynamic strains in the DPD andcompaction-induced prestrain by the stent were compared with leafletsexposed to compaction-induced prestrain only. The latter leaflets werecultured in the DPD with low-speed medium circulation withoutapplication of a dynamic pressure difference. As controls, unloadedrectangular-shaped valve leaflet tissue-equivalents, in which compactionwas not constrained, were used. The valve scaffolds were prepared from anon-woven PGA fiber mesh, coated with P4HB. Fibrin was used as a carrierfor the human saphenous vein cells to ensure homogeneous celldistribution throughout the scaffold and to render a compact structuresuitable for mechanical conditioning (Mol et al., 2005). The levels ofcompaction induced prestrain were estimated, as well as the straindistribution in the leaflets resulting from the dynamic transvalvularpressure applied in the DPD, the latter using numerical analyses.

In one embodiment the restricted or zero flow passage is at least 20%,preferably 50% or more, smaller than the flow passage of the finishedprosthesis. A dynamic pressure pulse creates more strain on the 3Dscaffold or developing tissue if a compensation flow through the flowpassage is kept to a minimum or zero. In case the restriction is formedby means of a coaptation area on the individual leaflets the flowpassage is chosen sufficient to either frequently of occasionally openthe valve.

In a further variant, the at least one perfusion flow is less than 50ml/min, preferably less that 5 ml/min. The perfusion flow is kept to aminimum and the opening in the 3D scaffold and/or developing tissueshould only be sufficiently sized to allow this small perfusion flowthrough it and only having few influence on the creation of the pressuredifference.

It is of further advantage if the at least one perfusion flow hassubstantially no pulsation. Especially the mimic of the normal flowbehaviour through the prosthesis should not be imitated. In case aroller pump is used, only the influence of the usual operation of thispump is present and no additional pulsation. The flow should be keptsubstantially constant.

In a further embodiment the pressure difference is substantially zero atthe beginning, subsequently increasing up to a mean peak pressuredifference and thereafter decreasing to the end of the duration of stayof the 3D scaffold and/or thereon developing tissue in the bioreactorchamber. First the tissue is given time to develop without substantialstrain. Then the mean distribution of strain is increased and afterreaching a mean peak pressure difference after a certain time itdecreases to the end of the duration of stay in the bioreactor.

A mean peak pressure difference, averaged for 24 hours is above 25 mmHg,preferably above 45 mmHg. This assures that a significant amount ofstrain is provided which enhances tissue formation and organisation.

Furthermore, the mean pressure difference could be, over a time period,which is 30-70%, preferably 45-55%, of the duration of stay in thebioreactor substantially zero. Such a time period gives tissuesufficient time for development on the 3D scaffold.

According to another embodiment the mean peak pressure differenceaveraged for 24 hours is reached after 60%, preferably after 70%, of theduration of stay in the bioreactor. As can be seen, the strain basedinfluence in the tissue formation and organisation is made in the secondhalf of the duration of stay in the bioreactor.

A modulated pressure difference could be used for a specific timeperiod, preferably 24 hours, to provide a respective strain distributionin that specific time period. The pressure difference could almost onlybe modulated with regard to a mean pressure difference per time periodbut also within the respective time period so as to gain a mean pressuredifference.

The dynamic pressure difference could have a frequency or 0.1-10 Hz,preferably 1 Hz. The dynamic pressure differences have a frequency,which is similar to an average heart sequence, but not the flow throughthe prosthesis is mimicked but the strain in the prosthesis is providedin this frequency.

The invention is further directed to a bioreactor for manufacturing atissue-engineered prosthesis having at least in an open condition a flowpassage, especially human heart valves, comprising:

-   -   A bioreactor chamber for inserting therein a seeded 3D scaffold,    -   at least one perfusion flow means to provide at least one        perfusion flow of a nutritient medium in the bioreactor chamber,    -   in addition pressurising means to apply a dynamic pressure        difference over the seeded 3D scaffold and/or thereon developing        tissue to create strain in the 3D scaffold and/or thereon        developing tissue, the pressuriser means having a compressible        and decompressible tubing in flow connection with the bioreactor        chamber.

The perfusion flow means and the additional pressurising means arepreferably independent from one another so that they apply a dynamicpressure difference overlaying the at least one perfusion flow whichusually has no pulsation. In order to not have a contamination of thenutrient medium a pressure pulse from the outside of the tubing isapplied. The tubing is compressible and returns back to its initialshape after pressure release.

In a preferred embodiment, at least a portion of the compressible anddecompressible tubing is placed in a cylinder surrounding the outersurface of said portion of the tubing and having a port for flow of acompressed fluid, preferably air, into the cylinder. The cylinder ispreferably manufactured from rigid material so that sufficient pressurecan be provided within the space between the cylinder and the tubing.

A magnet valve could be provided which is in flow communication with theport of the cylinder to control the flow of the compressed fluid intothe cylinder. The magnet valve could also control the outflow of thefluid out of the cylinder. A constant supply pressure could be providedwhich is modulated by the magnet valve so that within the cylinder thepredetermined pressure is present. This allows for an easy and costefficient modulation of the pressure.

Furthermore, a compliance chamber could be provided in flowcommunication with the bioreactor chamber to compensate for thedisplacement of the nutrient medium due to the pressure difference.Especially below the developing prosthesis some of the nutrient mediumis displaced. Due to only a minimum of perfusion flow, the displacedvolume of the nutrient volume has to be compensated by the compliancechamber. After pressure release the displaced nutrient medium flows backout of the compliance chamber and into the respective portion of thebioreactor chamber. The overall flow rate of the nutrition medium is notincreased.

In a further embodiment, the bioreactor chamber has a first and a secondportion each provided with a pressure sensor and holding means forholding the 3D scaffold and/or the thereon-developing tissue ispositioned between the first and second portion. With these two sensorsthe pressure difference from one side of the developing prosthesis tothe other side can be measured.

In the following, an embodiment of the present invention is described indetail. The following description should not, however, restrict theclaims. The claims should be understood in their broadest meaning. Thefigures show:

FIG. 1 a,1 b: Photographs of the valve scaffold, bottomview (a) andtopview (b).

FIG. 2 a,2 b: Schematic drawing of one DPD and its function (a) and aphotograph of six DPD systems in use simultaneously (b).

FIG. 3: Finite element mesh of the stented valve geometry. Because ofsymmetry only ½ of a leaflet is used in the finite element analyses.This part of the geometry is discretized using 224 hexahedral elements.

FIG. 4: A graph of the metabolic activity of cells cultured with thefirst four changes of the medium that has circulated in the DPD duringthe sterility test.

FIG. 5: A representative transvalvular pressure curve measured duringculturing of the valves.

FIG. 6 a,6 b,6 c,6 d.6 e.6 f: Representative sections of the valveleaflet tissues after four weeks of culturing stained with H&E (a,b,c)and Trichrome Masson (d,e,f).

FIG. 7 a,7 b: Stress-strain curves after four weeks of culturing ofnon-loaded leaflet tissue-equivalents, leaflets exposed to prestrain,and leaflets exposed to additional dynamic strains (a) and the evolutionof mechanical properties over time for dynamically strained leaflets(b).

FIG. 8 a,8 b,8 c: A graph of the dynamic strain distribution after fourweeks of culturing at the upstream (a) and downstream (b) surfaces ofthe leaflets at an applied dynamic transvalvular pressure difference of37 mm Hg. The grayscale represents the amount of dynamic strains in %.The relative amount of the overall dynamic strains found in the leafletand the mean value are shown in the histogram (c).

PREPARATION OF THE LEAFLET TISSUES The Heart Valve Scaffold

Trileaflet heart valve scaffolds were fabricated on a Fastacryl R°stent. The two components, Fastacryl powder and fluid (PMMA and MMMA,Vertex-dental, the Netherlands) were mixed, poured into a mold, andallowed to polymerize for 30 minutes. After complete polymerization, thestent was released from the mold. Anatomically shaped leaflets,including coaptation areas, were cut out of non-woven polyglycolic acidmeshes (PGA; thickness 1.0 mm; specific gravity 69 mg/cm³; Cellon,Luxembourg). The leaflets were coated with a thin layer ofpoly-4-hydroxybutyrate (P4HB; MW 1×10⁶; TEPHA Inc., Cambridge, USA) asdescribed before (Hoerstrup et al., 2000a). Before evaporation of thesolvent, the leaflets were positioned onto a teflon mold in the shape ofa trileaflet heart valve. The fastacryl stent was placed on top. Byaction of the solvent, dissolving the surface layer of the stent, theleaflets were fixed to the stent. After evaporation of the solvent, thevalve scaffold including the stent was removed from the mold (FIG. 1 a,1b). The valve scaffolds were further dried under vacuum overnight toremove solvent remnants, after which they were sterilized using ethyleneoxide. The leaflets contain coaptation to ensure closure of the valve.

Seeding Procedure

Cells harvested from the human vena saphena magna and expanded usingregular cell culture methods were used (Schnell et al., 2001). Themedium to culture these cells consisted of DMEM Advanced (Gibco, USA),supplemented with 10% Fetal Bovine Serum (FBS; PAN Biotech, Germany), 1%GlutaMax (Gibco, USA), and 0.1% gentamycin (PAN Biotech, Germany). Themedium used for seeding and subsequent tissue culture contained 0.3%gentamycin and additional L-ascorbic acid 2-phosphate (0.25 mg/ml;Sigma, USA) to promote extracellular matrix production. The scaffoldswere placed in medium overnight before seeding to facilitate cellattachment by deposition of proteins. The seeding was performed perleaflet using fibrin as a cell carrier (Mol et al., 2005). Briefly, thecells were suspended in a sterile thrombin solution (10 IU/ml medium;Sigma, USA) in a volume that equals half the void volume of the scaffold(0.5×length×width×thickness). The cells in thrombin were mixed with anequal amount of sterile fibrinogen solution (10 mg actual protein/mlmedium; Sigma, USA) and dripped onto the scaffold. The fibrin solutionwas taken up by the scaffold and remained inside due to polymerizationof the fibrin gel. The leaflets were seeded with a density of 4-5million cells (passage 6-7) per cm2 of scaffold. The seeded valvescaffolds were allowed to polymerize for 20 minutes in an incubator(37±C and 5% CO2) before placement into the DPD.

The Diastolic Pulse Duplicator (DPD) Description of the DPD

One DPD consists of a bioreactor [A], in which the valve is cultured,and a medium container [B]. They are connected to each other via twoparallel tubing series [C], which run through a roller pump [D]. Part ofthe upper tubing consists of a thicker tube placed in a polycarbonatecylinder [E]. Compressed air can be released into this cylinder, via amagnet valve [F], to compress and decompress the tube, resulting in apressure difference over the leaflets. A syringe [G], placed on thebioreactor, serves as a compliance chamber. The pressures upstream anddownstream of the leaflets is monitored using pressure sensors [H].

Each DPD consists of two components as shown in FIG. 2 (a): a bioreactor(height=9 cm, diameter=6 cm), in which the valve is cultured, and amedium container of similar dimensions, both fabricated frompolycarbonate (KUBRA Kunststoffen, The Netherlands). The bioreactoritself consists of two parts, the upper part containing a glass window(Melles Griot BV, The Netherlands) for visualization of the valve, and alower part, which can be screwed together. Silicone rings (van derHeijden, The Netherlands) were used to seal all components. Thebioreactor and the medium container are connected via two parallelsilicone tubing series (Rubber, the Netherlands). Polypropyleneconnector parts (Neolab, Milispec Int., The Netherlands) were used tosecure the tubing. Both tubing series run through a roller pump(Master°exr, ColeParmer, USA). Part of the upper tubing serie consistsof a thicker silicone tube placed in a polycarbonate cylinder, with aconnection to tubing suitable to withstand air under pressure (Festo,The Netherlands). This compressed-air tubing is connected to acompressed-air tap (7 bar). The air pressure is reduced to 2 bar andruns through a proportional magnet valve (Festo, The Netherlands) intothe polycarbonate cylinder. The complete DPD is sterilized by ethyleneoxide and is placed inside an incubator, together with the roller pump.Up to six DPDs can be placed onto one shelf of a normal sized incubatoras shown in FIG. 2 (b).

Functioning of the DPD

About 75 ml of medium is circulating from the medium container, throughthe bioreactor, and back into the medium container, via the roller pumpat very low speed (4 ml/min) to supply fresh nutrients to and to removewaste products from the developing valve tissue. A sterile filter (0.2μm, Schleicher & Schuell Bioscience, Germany) is placed in the lid ofthe medium container to oxygenate the circulating medium. About 25 ml ofmedium is present in the bioreactor and the tubing series, and theremaining 50 ml is present in the medium container and can be changedeasily. To apply a dynamic pressure difference over the leaflets, thesilicone tube in the polycarbonate cylinder is compressed anddecompressed by the air coming from the proportional magnet valve. Thetransvalvular pressure generated over the valve leaflets is controlledvia a programmable multi-10-card using LabView software (NationalInstruments, USA). The shape and frequency of the pressure wave isprogrammed for an optimal tranvalvular pressure. A syringe, connected tothe lower part of the biore actor, filled with 10 ml of medium and 40 mlof air, serves as compliance chamber. Pressure sensors (BD, Belgium),connected to both the lower and upper part of the bioreactor, are usedto measure the pressure upstream (at the ventricular side) anddownstream (at the arterial side) of the leaflets. Using the samemulti-10-card and software, the dynamic pressure difference could bemonitored and logged.

Sterility and Biocompatibility

To prove maintained sterility over several weeks, the DPD was sterilizedby ethylene oxide and filled with 75 ml of culture medium without theaddition of gentamycin. The system was fully functional for a period ofthree weeks. During the testing period, a culture flask filled withculture medium, without gentamycin, served as control. The medium in themedium container was changed every 3-4 days as well as two thirds of themedium in the culture flask. The medium was checked microscopically forcontamination and was stored at −20° C. until further use.

In order to ensure that the materials used in the DPD are suitable forcultivation of heart valves and that no toxic components are beingreleased, a biocompatibility test was performed using the medium thatwas stored during the sterility test. Human saphenous vein cells wereseeded into 24 wells plates (30.000 cells/well) and left overnight toattach and spread in an incubator in normal cell culture medium. Thenext day, the normal cell culture medium was replaced by the medium ofthe sterility test that had circulated through the DPD and the controlmedium (five wells per test group). The medium stored after the firstfour medium changes, as well as the control medium from the cultureflask, was used. The cells were allowed to grow for three subsequentdays, after which the metabolic activity of the cells, as a measure forviability, was determined using an MTT test (Mosmann, 1983). Briefly,MTT (Sigma, USA) in solution (5 mg/ml in PBS) was diluted in medium andadded to the cells. After one hour of incubation at 37±C and 5% CO2,metabolic active cells had converted the MTT salt into purple crystalslocated inside the cells. Isopropanol (VWR International, USA),containing 10% formic acid (Sigma, USA) was added to the cells torelease and dissolve the purple crystals and the optical density of thesolution was determined. The results are expressed as percentages withrespect to the control, which was set at 100%.

Tissue Culture and Mechanical Conditioning

Three experimental groups of valve leaflet tissues were engineered tocompare tissue formation and to show the feasibility of the strain-basedapproach. The first group comprises rectangular shaped valve leaflettissue-equivalents. They were cultured statically in a cell cultureflask for up to four weeks and could compact freely, serving asnon-loaded controls. The second and third group consisted of valveleaflets, all cultured in the DPD. Directly after placement in the DPD,all leaflets were exposed to medium circulation at low speed (4 ml/min)to supply fresh nutrients to the developing tissue. Valve leaflets werecultured up to four weeks, exposed to continuous medium circulation andprestrain due to compaction constrained by the stent (group 2).Additional valve leaflets were exposed to dynamic strains at 1 Hz inaddition to the prestrain and continuous medium circulation (group 3).The pressures upstream and downstream of the valves, as well as thedynamic transvalvular pressure, were recorded every three hours. Thevalve leaflets of group 3 were cultured up to two, three and four weeks.The medium in the medium container, as well as two thirds of the mediumin the culture flask, for group 1, was replaced every three to fourdays.

Evaluation of Tissue Formation Qualitative Tissue Analysis

Tissue formation in all groups was analyzed by histology after fourweeks. Representative pieces were fixed in phosphate-buffered formalin(Fluke, USA) and embedded in paraffin. Sections were cut at 5 ¹mthickness and studied by Haematoxylin and Eosin (H&E) staining forgeneral tissue morphology and Trichrome Masson staining for collagenformation, as collagen is the main load-bearing component of theextracellular matrix.

Mechanical Testing

The mechanical properties of circumferential strips of the engineeredleaflet tissues of the three groups after four weeks of culturing weredetermined by uniaxial tensile tests. As thickness measurements of thefresh strips are practically difficult, the thickness was determinedfrom representative histology sections. Stress-strain curves wereobtained using an uniaxial tensile tester (Instron, Belgium, model 4411,equipped with a load cell of 10 N) with a constant strain rate of 1.7%per second. To get insight into the evolution of mechanical propertiesin time, the mechanical properties of circumferential strips ofdynamically strained leaflets after two and three weeks of culturingwere additionally determined.

Estimation of Strains in the Leaflets

To estimate the amount of prestrain in the leaflets, due to compactionconstrained by the stent, the difference in size of the leaflets in thestent after culturing and after release from the stent was determined.The leaflets, having an initially bulged shape in the stent, didstraighten during culturing due to free compaction of the neo-tissue.The initial circumferential size of the leaflets was 23 mm, whereas infully straightened leaflets this size was reduced to 20 mm, indicatingthat the leaflets could compact freely up to 13% during culturing. Thecircumferential size of the leaflets was measured after release of theleaflets from the stent of the valves exposed to prestrain and mediumcirculation after four weeks. This measured size was divided by themaximally straightened size of 20 mm to calculate the amount ofprestrain in the leaflets. This value was subsequently extracted from 1and multiplied by 100% to obtain the amount expressed in a percentage ofprestrain.

Finite element analyses were used to estimate the amount anddistribution of the dynamic strains in the leaflets resulting from theapplied dynamic transvalvular pressure. The finite element mesh of thestented valve geometry is shown in FIG. 3. The configuration shown inthis figure was assumed to be stress-free and because of symmetry only ½of a valve leaflet was used in the finite element analyses. At thesymmetry edge, nodal displacements in the normal direction weresuppressed, whereas at the fixed edge all nodal displacements were setto zero. At the free edge, a contact surface was defined to modelcoaptation of adjacent leaflets. The transvalvular pressure ptv wassubsequently applied to the downstream surface of the leaflets. In thisstudy, the finite element package SEPRAN was used (Segal, 1984).

The valve tissues were modeled as an incompressible generalizedNeo-Hookean material:

σ=−pl+G(B−l)  (1.1)

with σ the Cauchy stress, p the hydrostatic pressure, l the unity tensorand G the shear modulus of the material. The left Cauchy-Greendeformation tensor was defined as B=F·F^(T) with F the deformationgradient tensor. To describe the (potential) non-linear behavior of theleaflet tissues, the following expression for G was used:

G=G0(l ₁(B)/3)^(n)  (1.2)

with G0 and n material parameters and l₁(B)=trace(B) the first invariantof B. The parameter of n is used to control the degree of non-linearity:n>0 results in strain hardening, n<0 in strain stiffening and for n=0the classical Neo-Hookean model is obtained. The material parameterswere obtained by fitting the constitutive law (Eq. 1.1) to the meanresults before failure of the uniaxial tensile tests of the leaflettissues exposed to dynamic strains after two, three, and four weeks ofculturing.

Results The DPD

The tested medium (dark gray bars) was compared to control medium (lightgray bars), which were set at 100%. The metabolic activity was in allcases 80-100% compared to the controls and, therefore, the DPD can beconsidered as being biocompatible. The error bars represent the standarderror of the mean.

The medium, without addition of antibiotics, that had circulated in theDPD did not show any macroscopic or microscopic signs of contaminationfor the complete test period. The system was easy to handle and the riskof contamination during medium replacement was minimal. The results ofthe biocompatibility test, performed for the first four medium changesduring the sterility test, are shown in FIG. 4. The metabolic activityof the cells cultured with the four tested media were all within 80 to100% when compared to the controls, indicating the DPD to bebiocompatible and suitable for cultivation of human heart valves.

Dynamic Straining Protocol

A representative transvalvular pressure curve, measured during culturingis shown in FIG. 5. The definitions of the DC offset, the cyclictransvalvular pressure difference, and the dynamic transvalvularpressure difference are represented. The permanent transvalvularpressure, present over the valve leaflets, is referred to as the DCoffset, which is the average value of the difference in pressureupstream and downstream of the valve. The cyclic transvalvular pressureis defined as the peak-to-peak transvalvular pressure value. The maximumtrans-valvular pressure value, including the DC offset and the cyclictransvalvular pressure, is referred to as the applied dynamictransvalvular pressure.

The dynamic transvalvular pressure was averaged for each day, resultingin dynamic trans-valvular pressures increasing gradually from 0 to about80 mm Hg within the first two weeks of culturing. The last two weeks ofculturing, the pressures were lowered, due to the expected loss of thesupport function of the scaffold, and kept constant at about 37 mm Hg.

Evaluation of Tissue Formation Histology

Sections stained with H&E and Trichrome Masson (FIG. 6) showed superiortissue formation in the leaflets (FIG. 6( a), 6(b), 6(d), 6(e)) whencompared to the non-loaded rectangular leaflet tissue-equivalents (FIG.6( c), 6(f)). The tissue of the leaflets cultured with additionaldynamic strains (FIG. 6( a), 5.6(d)) appeared to be more homogeneous anddenser when compared to the leaflets exposed to prestrain only (FIG. 6(b), 6(e)). Collagen could be identified in the leaflets, either culturedusing prestrain or additional dynamic strains, after four weeks ofculturing (FIG. 6( e), 6(d)). The bars in the images represent scales of350 ¹m. The dynamically strained leaflets (a,d) and the leaflets exposedto prestrain only (b,e) showed superior tissue development when comparedto the non-loaded valve leaflet tissue-equivalents (c,f). Thedynamically strained leaflet tissue seemed more homogeneous and denseras compared to the leaflets exposed to prestrain only. Collagen, stainedblue, could be identified in the leaflets (d,e).

Mechanical Tests

Representative stress-strain curves for all groups after four weeks ofculturing are shown in FIG. 7( a). The non-loaded leaflet equivalentsshowed linear behavior, while the leaflets, exposed tocompaction-induced prestrain and either with or without additionaldynamic strains, showed more tissue-like non-linear behavior after fourweeks of culturing (a) The evolution of mechanical properties withincreasing culture time is shown in FIG. 7( b) for the leaflets exposedto dynamic strains. After two weeks of culturing, the mechanicalbehavior was linear, representing scaffold-like behavior. After threeand four weeks, the tissue showed more non-linear mechanical behavior,representative for tissue contribution.

Estimation of Strains in the Leaflets Prestrain

The amount of prestrain in the leaflets of the valves exposed toprestrain only after four weeks of culturing, assuming maximalstraightening of the leaflets, was shown to vary between 3 and 5%.

Dynamic Strains

The constitutive law (Eq. 1.1) fitted the results of the uniaxialtensile tests quite reasonable. Table 1.1 summarizes the inputparameters for the finite element model, the mean absolute error of thefits, and the estimated mean dynamic strains after two, three and fourweeks of culturing, based on a transvalvular pressure difference of 37mm Hg. The mean dynamic strains in the leaflets increased from 8% aftertwo weeks to about 20% after four weeks of culturing. The dynamic straindistribution in the leaflets after four weeks of culturing is shown inFIG. 8 a,8 b,8 c, with in FIGS. 8 (a) and 8(b) the strain distributionwithin one leaflet at respectively the upstream and downstream surfaceand in FIG. 8( c) the estimated range of overall dynamic strains withina leaflet.

TABLE 1.1 Summary of the input parameters for the finite elementanalyses of the dynamically strained leaflets after two, three, and fourweeks of culturing. The parameter n has been set to zero for theleaflets after two weeks of culturing, due to the observed linearbehavior. Furthermore, the mean absolute errors of the fits of theconstitutive law (Eq. 1.1) to the results of the uniaxial tensile testsand the resulting estimated mean dynamic strains are represented. MeanCulture time Thickness G₀ n absolute error Mean dynamic [weeks] [mm][kPa] [—] of fit [kPa] strain [%] 2 0.80 188 0.0 0.95 8 3 0.59 51 8.63.93 24 4 0.63 59 9.2 4.61 20

DISCUSSION

Mechanical stimulation of tissue formation is a well-known technique intissue engineering of cardiovascular structures to improve tissueformation and organization. Various conditioning approaches are beingemployed in bioreactor systems, from flow-based to strain-based to evenmimicking the exact physiological environment in the body. For tissueengineering of blood vessels, flow-based as well as strain-basedapproaches are used, however, for tissue engineering of heart valves,only flow-based approaches have been described. The optimal conditioningprotocol depends on several factors, such as the sensitivity of the cellpheno-type and source (i.e. animal or human) to mechanical cues, thescaffold used, the transfer of the mechanical cues from the scaffold tothe cells, and the magnitude and type of mechanical cue.

If the forms was put on tissue engineering of human heart valve leafletsengineered from human saphenous vein cells seeded using fibrin as a cellcarrier onto PGA/P4HB trileaflet heart valve scaffolds (FIG. 1 a,1 b).For this particular cell type, being more sensitive to mechanicalstimulation as compared to human arterial derived cells, we previouslyhave shown a large impact of cyclic straining during culturing on tissueformation (Mol et al., 2003). In this former study, the amount ofextracellular matrix formation increased when larger strains were used.Flow is most likely not necessitated in the early phase of tissuedevelopment, but might start to play a significant role as soon as theleaflets are to be seeded with endothelial cells in a later phase. Flowmight then stabilize the tissue via signaling by the endothelial cellsand prepare the tissue for subsequent implantation. This study describesa strain-based approach to tissue engineer human heart valve leaflets.In order to expose the developing heart valve leaflets to dynamicstrains, a pressure difference had to be applied over the leaflets,mimicking the diastolic phase in the heart. A novel bioreactor, theDiastolic Pulse Duplicator (FIG. 2 a,2 b), is developed for thispurpose, which can expose the developing tissue to increasing amounts ofdynamic strains. Besides application of dynamic strains, the leafletscultured in this study were exposed to prestrain, due to tissuecompaction constrained by the stented geometry. The flow was kept low (4ml/min) and served solely to provide the tissue with sufficient freshnutrients and to remove waste products. The DPD was very easy to handle,sized small with a total medium volume of only 75 ml per valve, provento be biocompatible (FIG. 4), and sterility could be maintained overprolonged periods of time.

Rectangular non-loaded valve leaflet tissue-equivalents were shown torender much less tissue formation after four weeks of culturing whencompared to the superior tissue formation in the leaflets, exposed toeither prestrain only as well as to additional dynamic strains (FIG. 6).The tissue of the leaflets exposed to dynamic strains appeared to bemore homogeneous and denser packed as compared to leaflets exposed toprestrain only, however, this concerned only qualitative observations byhistology. The mechanical properties (FIG. 7 a,7 b) of the leafletsexposed to dynamic strains showed increased non-linear tissue-likebehavior over time, indicating increasing amounts of tissue andcollagen. After four weeks of culturing, the non-loaded rectangularshaped leaflet tissue-equivalents showed linear behavior, representativefor scaffold behavior. The leaflets, exposed to prestrain alone as wellas to additional dynamic strains showed non-linear behavior after fourweeks of culturing, correlating with the larger amounts of tissue foundin the leaflets as compared to the non-loaded leaflettissue-equivalents. The continuous medium circulation in the DPD willmost likely contribute to the improved tissue formation in the leafletsas well.

Prestrain alone obviously resulted in abundant amounts of tissue. Theprestrain is estimated to be in the range of 3 to 5% and might alreadybe sufficient for optimized tissue formation. However, dynamic strainingmight further enhance tissue organization and, furthermore, mightrepresent a valuable tool to maintain the bulged shape of the leafletsby some deformation of the scaffold and the tissue. Furthermore, forfuture applications towards tissue engineering of stentless human heartvalves, dynamic strains will be of larger importance as the prestrainsin stentless valves will be much less.

To get insight into the amount of dynamic strains applied to theleaflets, the dynamic strain distribution was estimated. The materialproperties of the leaflets determine the amount of dynamic strains at agiven dynamic transvalvular pressure. The transvalvular pressure wasmonitored in the DPD during culturing (FIG. 5). Theoretically, theamount of dynamic strains can be monitored using markers on the leafletsand subsequent imaging of the leaflets being strained, followed by imageanalyses. As this is a time-consuming and in practice rather difficultmethod, we have chosen to estimate the dynamic strains in the leafletsby finite element analyses. The material parameters of the developingleaflets, determined by fitting the constitutive law (Eq. 1.1) to themean results of the uniaxial tensile tests before failure, were used toserve as input for the finite element analyses. The mean dynamic strainin the valve leaflets cultured in this study, as an example for futureuse, were estimated to vary from 8% at two weeks to 20% at four weeks ofculturing (FIG. 8 a,8 b,8 c and Table 1.1). The estimated dynamicstrains increased between week two and three, while the applied dynamictransvalvular pressures were similar, indicating loss of scaffoldsupport, resulting in a decreased stiffness of the tissues, as shown bya lower shear modulus after three weeks of culturing. For the stentedvalve geometry, the amount of applied dynamic strains in this studymight be too large for optimized tissue formation, when taking theprestrain into account, but application of large dynamic strains in theDPD was shown feasible. As currently the strains in the leaflets aredetermined afterwards, a future research focus for optimization of theDiastolic Pulse Duplicator is the development of a non-invasive methodto determine tissue strains directly during culturing and to integratethis feature in a feedback loop to control the magnitude of the strains.

In the finite element analysis, an initial stress-free configuration ofthe leaflets is assumed, which is not completely true as the valveleaflets show compaction during culturing. The material properties ofthe leaflets are assumed to be homogeneous and isotropic, which mightnot be the case after a certain culture period due to the influence ofprestrain in mainly the circumferential direction and the local dynamicstrain distribution in the leaflets. Therefore, leaflets to be culturedin the DPD will be mechanically tested in both circumferential andradial direction to identify possible anisotropic properties. In case ofanisotropic properties, an extended model should be used for the finiteelement analyses as described by Driessen et al. (2004).

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1.-16. (canceled)
 17. A bioreactor for manufacturing a tissue-engineeredprosthesis having at least in an open condition a flow passage,especially human heart valves, comprising: a bioreactor chamber forinserting therein a seeded 3D scaffold, at least one perfusion flowmeans to provide at least one perfusion flow of a nutrient medium in thebioreactor chamber, in addition pressuriser means to apply a dynamicpressure difference over the seeded 3D scaffold and/or thereondeveloping tissue to create strain in the 3D scaffold and/or thereondeveloping tissue, the pressuriser means having compressible anddecompressive tubing in flow connection with the bioreactor chamber. 18.The bioreactor of claim 17, wherein at least a portion of thecompressible and decompressible tubing is placed in a cylindersurrounding the outer surface of said portion of the tubing and having aport for flow of a compressed fluid, preferably air, into the cylinder.19. The bioreactor of claim 18, wherein a magnet valve is provided beingin flow communication with the port of the cylinder to control the flowof the compressed fluid into the cylinder.
 20. The bioreactor of claim17, wherein a compliance chamber is provided in flow communication withthe bioreactor chamber to compensate for the displacement of thenutrient medium due to the pressure difference.
 21. The bioreactor ofclaim 17, wherein the bioreactor chamber has a first and a secondportion each provided with a pressure sensor and holding means forholding a 3D scaffold and/or the thereon developing tissue is positionedbetween the first and second portion.
 22. A method of manufacturing atissue-engineered prosthesis having at least in an open condition a flowpassage, especially human heart valves, comprising the steps of: placinga seeded 3D scaffold in a bioreactor chamber, providing at least oneperfusion flow of a nutrient medium in the bioreactor chamber to supplysaid seeded 3D scaffold and/or a thereon developing tissue withnutrients whereby a flow passage of the seeded 3D scaffold and/or thethereon developing tissue in relation to the flow passage of thefinished prosthesis is restricted or zero, applying in addition adynamic pressure difference over the 3D scaffold and/or thereondeveloping tissue depending on the condition and/or stage of tissuedevelopment to create strain in the 3D scaffold and/or thereondeveloping tissue, and opening the restricted or zero flow passage toits finished size.
 23. The method of claim 22, wherein the prosthesis isa heart valve having leaflets and the restriction is achieved bycoaptation of the leaflets.
 24. The method of claim 22, wherein therestricted or zero flow passage is at least 20%, preferably 50%, smallerthan the flow passage of the finished prosthesis.
 25. The method ofclaim 22, wherein the at least one perfusion flow is less than 50ml/min, preferably less than 5 ml/min.
 26. The method of claim 22,wherein the at least one perfusion flow has substantially no pulsation.27. The method of claim 22, wherein the pressure difference issubstantially zero at the beginning, subsequently increasing up to amean peak pressure difference and thereafter decreasing to the end ofthe duration of stay of the 3D scaffold and/or thereon developing tissuein the bioreactor chamber.
 28. The method of claim 22, wherein a meanpeak pressure difference averaged for 24 hours, is above 25 mmHg,preferably above 45 mmHg.
 29. The method of claim 22, wherein the meanpressure difference over a time period, which is 30-70%, preferably45-55%, of the duration of stay in the bioreactor, is substantiallyzero.
 30. The method of claim 29, wherein the mean peak pressuredifference averaged for 24 hours is reached after 60%, preferably after70%, of the duration of stay in the bioreactor.
 31. The method of claim30, wherein the modulated pressure differences per time period,preferably 24 hours, are used to provide respective strain distributionin that time period.
 32. method of claim 22, wherein the dynamicpressure difference has a frequency of 0.1 to 10 Hz, preferably 1 Hz.